How to add type IIS restriction site sequence in primer for PCR amplification by using snapgene?
I do not know how to use snapgene but you design the primers using whatever software you like and these primers will anneal to the template and give you your pcr annealing conditions. Then you add the cut site at the 5' end of each primer and then add 3 random bases at the 5' end of the primer with cut site . These extra bases are needed for enzyme binding before the enzyme cuts. Then you run your pcr using annealing only for the template specific part of the primers.Do not use the whole sequence plus enzyme site annealing temperature because in the first few cycles there is no template including these sequences. If the cut site is downstream by a known number of bases then sequence will have to be added appropriate to where the enzyme cuts not where it binds
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